Post-translational modifications of endothelial nitric oxide synthase (eNOS) play a very important part in regulating eNOS activity, endothelial nitric oxide (NO) production, endothelium-dependent vasorelaxation, and vascular tone. The roles of phosphorylation, acylation, and glycosylation in regulating eNOS activity are well recognized. However, the importance of acetylation, as a post-translational modification, in governing eNOS activity has not been carefully studied. Based on novel preliminary evidence, this application proposes that eNOS is acetylated in the endothelium, and site-specific de-acetylation of eNOS has an important role in governing eNOS activity, endothelium-dependent vasorelaxation, and vascular tone. We suggest that the ubiquitously expressed protein deacetylase SIRT1 (Silent Information RegulaTor), by de-acetylating eNOS at specific acetylated lysine residues, stimulates eNOS activity, endothelial NO production, and promotes endothelium-dependent vasorelaxation. This application will examine if eNOS is a direct target of SIRT1, and characterize in detail the mechanism through which SIRT1 stimulates eNOS activity. The importance of a SIRT1-dependent mechanism in governing endothelium-dependent vascular relaxation, and the mediating role of eNOS in this mechanism, will be assessed. The in vivo relevance of SIRT1 with respect to modulation of physiologic changes in vascular tone will be determined. Finally, the regulation of human SIRT1 expression by a redox-sensitive transcriptional mechanism involving the p53 transcription factor and its transcriptional co-activator redox factor-1, and the physiologic importance of this transcriptional mechanism in regulating vascular SIRT1 expression, will be explored. In summary, this application will determine if eNOS is a target of the SIRT1 deacetylase, and in doing so, promises to define a novel mechanism for the regulation of endothelium-dependent vascular relaxation. It will also explore the physiologic importance of this mechanism in controlling vascular tone in vivo. As such, it may offer new targets and strategies for the treatment of human disorders of endothelial function and dysregulation of vascular tone. PUBLIC HEALTH RELEVANCE Production of nitric oxide in blood vessels is a very important means for the regulation of blood vessel function. This project examines a novel means by which production of nitric oxide in blood vessels is controlled. Understanding this new mechanism that controls nitric oxide in blood vessels will help further our knowledge about how blood vessels in disease states such as high blood pressure and atherosclerosis have lower levels of nitric oxide.